二点螟几丁质酶1基因CiChi1的克隆和功能分析

Cloning and functional analysis of chitinase 1 gene CiChi1 in yellow top borer Chilo infuscatellus

为明确几丁质酶1(chitinase 1,Chi1)编码基因在二点螟Chilo infuscatellus中的潜在功能,对Chi1基因进行全长序列克隆和生物信息学分析,使用实时荧光定量PCR技术检测该基因的时空表达模式,利用RNA干扰技术探究其在二点螟生长发育方面的功能。结果表明,二化螟Chi1基因序列全长为1788 bp,开放阅读框为1653 bp,编码550个氨基酸,并命名为CiChi1;CiChi1基因在二点螟不同发育阶段及不同组织中均有表达,其中在成虫期的相对表达量最高,在6龄幼虫期的相对表达量次之,且在6龄幼虫表皮中的相对表达量最高;在RNA干扰试验中,注射dsCiChi1的二点螟3龄幼虫试验组在第14天的最终死亡率为62.0%,显著高于注射dsEGFP的阴性对照组(27.8%)和注射蒸馏水的空白对照组(33.9%);此时试验组的平均虫重为0.024 g,显著低于阴性对照组(0.039 g)和空白对照组(0.040 g),且试验组试虫还伴随有外表皮急剧变黑、消解软化的致死表型;实时荧光定量PCR检测结果表明,试验组试虫体内CiChi1基因表达量较阴性对照组和空白对照组显著下调。表明CiChi1基因可能主要参与二点螟变态发育时期和成虫时期的形态建成,同时调控二点螟幼虫阶段表皮的再生与消解。

In order to dissect the function of chitinase 1(Chi1)gene of yellow top borer Chilo infuscatellus,the full length of Chi1 was cloned and analyzed by bioinformatics method.In addition,the spatio-temporal expression pattern of Chi1 was detected by quantitative real-time PCR and RNA interference was used to explore its potential function in growth and development.The results showed that the cDNA of Chi1 gene was 1788 bp in length with an open reading frame of 1653 bp encoding 550 amino acid residues,which was named as CiChi1.CiChi1 was expressed during all developmental stages and all tissues,with the highest expression in adults,followed by 6th-instar larva,and its highest expression was detected in epidermis.In the RNA interference experiment,the average mortality rate of the dsCiChi1 injection group was 62.0%at day 14,which was significantly higher than that of the negative control group injected with dsEGFP(27.8%)and the CK group injected with distilled water(33.9%).The average weight of the treatment group was 0.024 g,which was significantly lower than that of the negative control group(0.039 g)and the CK group(0.040 g),and the lethality of the dsCiChi1 group was accompanied by rapid blackening of epidermis and decomposition and softening of insect body.The results of real-time fluorescence quantitative PCR showed that the expression of CiChi1 gene in the treatment group was significantly lower than the negative control group and CK group.The results indicated that CiChi1 gene might be mainly involved in the morphogenesis during metamorphosis and adult stage,and regulating the regeneration and digestion of cuticle during larval stage.